Dataset Information

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii.

ABSTRACT:

Background

This study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.

Methods

Antimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdilution method. Moreover, the MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PA?N). Regulatory genes of the RND efflux pumps (adeRS, adeL, adeN and baeSR) were subjected to sequencing. The relative expression of adeB, adeG and adeJ genes was determined by quantitative real-time PCR (qRT-PCR).

Results

Overall, the majority of isolates (94%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of the isolates against multiple antibiotics mainly tigecycline. However, we found no efflux activity against imipenem. Several amino acid substitutions were detected in the products of regulatory genes; except in AdeN. Of note, G186V mutation in AdeS was found to be associated with overexpression of its efflux pump. No insertion sequences were detected.

Conclusions

Our findings outlined the role of RND efflux pumps in resistance of A. baumannii to multiple antibiotics particularly tigecycline, and pointed out the importance of a variety of single mutations in the corresponding regulatory systems. Further studies are required to decipher the precise role of RND efflux pumps in multidrug-resistant clinical isolates of A. baumannii.

SUBMITTER: Salehi B

PROVIDER: S-EPMC7944621 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Publications

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii.

Salehi Bahare B Ghalavand Zohreh Z Yadegar Abbas A Eslami Gita G

Antimicrobial resistance and infection control 20210310 1

<h4>Background</h4>This study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.<h4>Methods</h4>Antimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdil ...[more]

PMID: 33691788

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Project description:Overexpression of chromosomal resistance-nodulation-cell division (RND)-type efflux systems with broad substrate specificity contributes to multidrug resistance (MDR) in Acinetobacter baumannii We have shown that modulation of expression of the structural genes for the efflux systems AdeABC and AdeIJK confers MDR and results in numerous alterations of membrane-associated cellular functions, in particular biofilm formation. However, the contribution of these RND pumps to cell fitness and virulence has not yet been studied. The biological cost of an antibiotic resistance mechanism is a key parameter in determining its stability and dissemination. From an entirely sequenced susceptible clinical isolate, we have generated a set of isogenic derivatives having single point mutations resulting in overexpression of each efflux system or with every pump deleted by allelic replacement. We found that overproduction of the pumps results in a significant decrease in fitness of the bacterial host when measured by competition experiments in vitro Fitness and virulence were also evaluated in vivo both in systemic and pulmonary infection models in immunocompetent mice. A diminished competitiveness of the AdeABC-overexpressing mutant was observed only after intraperitoneal inoculation, but not after intranasal inoculation, the latter mimicking the most frequent type of human A. baumannii infection. However, in mice infected intranasally, this mutant was more virulent and stimulated an enhanced neutrophil activation in the lungs. Altogether, these data account for the observation that adeABC overexpression is common in MDR A. baumannii frequently found in ventilator-associated pneumonia.Overproduction of the RND AdeABC efflux system is observed with a high incidence in multidrug-resistant Acinetobacter baumannii and results in increased resistance to several antibiotics of choice for the treatment of infections caused by this nosocomial pathogen. It was therefore important to study the biological cost of the overexpression of the adeABC structural operon which is normally tightly regulated. Fitness diminution of an overexpressing mutant detected in vitro and in vivo in a model that mimics sepsis was not observed in a pulmonary infection model in which the mutant was more virulent. This points out that increased virulence can occur independently from prolonged persistence in the infected organ and can account for the elevated incidence of this resistance mechanism in clinical isolates. The study also indicates that transposon libraries will identify only virulence genes that are expressed under physiological conditions but not those that are tightly regulated.

Project description:Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with "experimentally verified," "presumed," and "probably irrelevant" degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the ISAba1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the "experimentally verified" level was permitted for 42 isolates to 7-8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria.

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Project description:Antibiotic-resistant Acinetobacter baumannii causes infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to the resistance-nodulation-cell division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps in A. baumannii ATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps of A. baumannii are tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to the overproduction of RND efflux pumps.IMPORTANCE RND-type efflux pumps are important contributors in development of clinical antibiotic resistance in A. baumannii However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed antibacterial activities of antibiotics in strains with different permeability barriers and found that the role of active efflux in antibiotic resistance of A. baumannii is limited to a few select antibiotics. Our results further show that the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates. Additional mechanisms of resistance, in particular those that improve the permeability barriers of bacterial cells and act synergistically with active efflux pumps are likely involved in antibiotic resistance of clinical A. baumannii isolates.

Dataset Information

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii.

Background

Methods

Results

Conclusions

Publications

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii.

Similar Datasets

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